Pregnancy antigen



Patented May I 26, 1936 PATENT OFFICE PREGNANCY ANTIGEN Gruskin,Philadelphia, Pa., assignor to Benjamin Inkeland Foundation, Chicago,111.,

tion of Illinois a corpora- No 1mm. Application June 18,1984,

- Serial m. 131,111

comm.

This'invention relates to 'a substance referred to as antigen.

, l This application is a continuation in part of application SerialNo.D3345, filed December The primary object of the present invention is toprovide an antigen for determining whether or not a fe'maleis in thestate of pregnancy.

Another object of the invention is the provision 1 of an antigenprepared from the placenta preferably human to determine whether or nota'female is pregnant.

Another object of the invention is a. test to 'determinewhether or not afemale human being is pregnanti'by an intradermal injection of an I 1antigen prepared from the'human placenta.

A still further object of the invention is the method ofpreparinga'n'antigen consisting in obtaining human placenta from themother with- 0 in such a short 'time as to preclude tissuedeterioration, preferably within two hours after birth, a and reducingthe placenta to a powder to which powder there are added severalingredients and chemicals to form the proper solution forperforming atest to determine whether or not a'female human is pregnant. I Anotherobject of the present invention is provide an antigen for intradermalinjection in a woman for the determination of'pregnancy. Numerous otherobjects and advantages will be apparent throughout the progress of thefollowing specification.

The theory upon which the present invention theory herein expounded, andof which innumerable test's have proven correct, an antigen made fromthe placenta, introduced into the skin of-a female who is pregnant, willcause the formation of pseudopodia, which will appear at the margi ofthe whea'l'formed by the injection.

' Antwan a The antigen is from the human or -mammalian placenta,- whichis obtained prefer-'- ably not later than two hours after birth becauseof the disintegration of the tissued cells after that time, it beingmade certain that the placenta is obtained mm a perfectly healthypatient. The

commonly producing pseudopodia. Therefore, upon the placenta so obtainedis thoroughly washed in running water, and the membranes and the fibrousportion cut into pieces of the desired size which may be about threeinches square to facilitate the washing of the placenta, so that alltraces of blood may be removed. When the blood has been thoroughlywashed away, the placental tissue is ground in a meat grinder. Theground placenta is then placed in acetone for dehydrating and forremoving lipoids, there being one part. ofv the tissue to four parts ofthe acetone. The-tissue and placenta are placed in tubes, and thoroughlyshaken in the acetone, and allowed to remain therein for five hours. Thetubes with the acetone and ground placenta are then centrifuged for fiveminutes, after which time the acetone is poured off, the cells remainingin the bottom of the tubes. The placental tissue or cells are thenremoved from the tubes and are spread out on fiat dishes to drythoroughly, and to permit rapid evaporation of the acetone. whenthoroughly dried, it is ground in a mortar to a fine powder. 1

The dry tissue of the placenta just described is extracted with aninorganic hydroxide, such as sodium hydroxide. One-tenth normal sodiumhydroxide (NaOH) in the proportion of one and one-half grams of theplacental tissue to one hundred cubic centimeters of sodium hydroxide,one-tenth normal is preferred. The sodium hydroxide, one-tenth'normal(NaOH') is made up 1 in the proportion of four grams of sodium.

hydroxide C. P. to one liter of distilled water in a volumetric flask.The ground' tissue is first ground to a smooth paste in a mortar byfirst adding a few cubic centimeters of the sodium hydroxide, and thenadding the rest of the onetenth normal sodium hydroxide slowly. Thecells are carefully mixed with the solution, so that a perfectly smoothsuspension of the cells in the sodium hydroxide is obtained. The extractof the cells and sodium hydroxide is then poured into large test tubesor a straight sided cylinder and allowed to stand for 24 hours, afterwhich time, the supernatant solution is pipetted off. The volume of thesupernatant solution should be measured in a sterilized graduate. Thissuperv natantsolution, which is the alkaline extract of the placentaltissue, is then placed in a sterile bottle of. a volumemore than twicethat of the extract so as' to allow for the addition of an acid andbuffer solution, which 'is used in neutralizing the alkaline extract. a

' The acid and buifer solution for neutralizing the alkaline extractcomprises 2.27 grams of anhydrous C. P. primary potassium phosphate(KHzPO4) and 4.235 cubic centimeters of concentrated hydrochloric acid,(HCl, 0. P.) (specific gravity 1.18-1.19, 35% solution) made up to oneliter with distilled water in a volumetric flask.

This gives a solution which is .0 5 normal with respect to primarypotassium phosphate, and which is .05 normal with respect tohydrochloric acid.

The acid andbuffer solution just described is added to the alkalineextract which has been pipetted oil. from the cells and measured. Theacid andbufler solution should be added slowly and the solutionscarefully stirred or gently agitated while the acid and buffersolutionis being added. After the acid and buffer solution has beenadded in an amount equal to the alkaline extract of the tissue. a fewmore cubic centimeters of the acid and buffer solution may be required.The resultant solution should then betested to see if the neutralizationis nearing theend point.

thvmol-blue as an indicator.

The standard solution is' made ofanhydrous primary-potassium phosphate,l/15 mqla'r, 9.078

grams of the puresalt, in one liter of freshly distilled water in avolumetric flask, and anhydrous secondary sodium phosphate, 1/15 molar,

' 9.472 grams of pure salt, made up to one liter with freshly distilledwater in a volumetric flask.

These solutions are combined in a pyrex conitainer in the proportion of4.9 parts of the solution of primary potassium phosphate to 5.1 parts ofthe solution of secondary sodium phosphate. The mixture of thesesolutions has a pH of 6.9.

When the end point of the titration has been reached, as ascertained bythe electrometric method, or by the matching of the antigen to thestandard solution of pH6,9, a preservative which consists of a solutionof tri-cresol and glycerin C. P.in the proportion of one part oftri-cresol to two parts of glycerin may be added.

The total volume of the finished. antigen is calculated, that is, thevolume including the alkaline extract and the added volume of the acidand buffer mixture. To each ten cubic centimeters of antigen there isadded two drops-of the tri-cresol and glycerin preservative from acapillary pipette having an internal diameter of one millimeter at thedropping end. A sterile stopper should be placed "on the bottle contaiiiing the antigen, and the solution should at once beshaken thoroughly sothat the preservative will be evenly dispersed throughout the antigen. Arubber stopper neutral in reaction and of the cap'typeis preferably usedfor the bottle containing the antigen so that the solution may be I the.antigen practically all of the placental tissuemay be utilized,including the maternal. foetal layers. gives a very to the laws ofprotein desensitization or allergy.

pyrex syringe and put into small pyrex vials for practical and definiteantigent for general uses and purposes. However, for the differentialdiagnosis between early and late pregnancy it has been found that in thelater months of pregnancy the foetal layer of placenta gives a plainerand strong,- 5 er reaction than when the whole placenta is used. Whilein early pregnancy ithas been found that the material layer of theplacenta gives a plainer and stronger reaction than if purely foetal orif mixed placenta were used. However, the mixture of both layers ispractical throughout entire pregnancy'and gives a plain and strongreaction capable for all practical intents and purposes. Of

course, proportions of the foetal and maternal layers may be varied tosuit the desired requirements. An antigen relating specifically to theuse Test One-tenth of one cubic'centimeter of the above antigen is drawnoff into a small syringe to which there is attached a very fine shortneedle. The antigen is injected intradermally, after first sterilizingand treating the surface of the patients skin and rendering it perfectlydry. The, injectionis performed by stretching the skin with one hand andinjecting the antigen intradermally, the injection being made by .theusual intradermal method. In positive cases, that is,.1 1 cases wherethe patient examined is pregnant, a slight area of reaction will benoticed surrounding the small bubble, termed a wheal, which wheal occursfrom the injection, and pseudopods will form. Pseudopods are radialelongations extending outwardly from the edges of the wheal. In negativecases, that is, in cases where the patient is not pregnant, no.pseudopod formation will take place. a i I The above test is based onthe fact that since theplacenta'is made up of both maternal and foetallayers, a characteristic protein of foetal embryonic character is partlyabsorbed into the system of the mother. when an extract made up from theplacental material is injected intradermally, Pseudopods will be formedbecause of the homologous nature of the protein, according The inventionand discovery herein set forth an designates, to a high degree ofcertainty, whether or not a female is pregnant. The particular antigenherein described is made from placenta than two horus after birthbecause of the disintegration of tissued cells, and while the exactmethod herein described is preferable, it is to be understood thatvarious changes, to a certain de- ,obtained from the mother preferablynot later 55 area, may bemade without departing from the spirit of theinvention or sacrificing any of its advanhles. and the rightishereby'reserv'ed to make all such changes as fairly fall within thescope of the followingclaims. i

The invention is hereby claimed as follows:

1. An antigen specific to the determination of pregnancy by intradermalinjection comprising a neutralized, inorganic alkaline hydroxide extractof placental tissue.

. 2. An antigen specific'to the determinationof 1o pregnancy byintradermal injection comprising a neutralized, inorganic alkalinehydroxide extract of placental tissue having 2. pl! of substantial:7.6-9- 3. to tho-determination of ll pregnancy by intradermal injectioncomprising a neutralized sodium hydroxide extract of mammalian placentaltissue.

4. An antigen specliic to the determination of pregnancy by intradermalinjection comprising a neutralized sodium hydroxide extract of humanplacental tissue having a pH of substantially 6.9.

5. The process of making an antigen for intradermal use to determine ifpregnancy exists which consists in extracting placental tissue with anirrorganlc alkaline hydroxide, separating the extract, and thenneutralizing the extract.

6. The process of making an antigen for intradermal use to determine 1!pregnancy exists which consists in obtaining an inorganic alkalineextract of placental tissue, and then adding an acid and builer solutionto reduce the extract to a pH of approximately 6.9.

7. The process of making an antigen for intraso dermal use to determineif pregnancy exists comprising extracting'placental tissue with sodiumhydroxide, separating the extract, and then neutralizing the extractwith potassium phosphate and hydrochloric acid.

8; The process oi making an antigen for intradermal use to determine ifpregnancy exists comprising extractinghuman placental tissue withone-tenth normal sodium hydroxide (NaOH) and then adding an acid andbutter solution oi'potass'ium phosphate (KHzPOO and hydrochloric acid(110]) to reduce the extract to a pH oi substantially 8.9.

9. An antigen to determine if pregnancy exists comprising an extract oimammalian placental tissue adapted for intradermal injection, whichextract contains a specific foetal embryonic protein homologous to thespecific foetal embryonic protein of a pregnant mammalian and whichproduces a skin reaction by pseudopod formation when the said antigen isinjected intradermally in a pregnant female mammalian. 20

BENJALHN GRUSKIN.

Patent No. 2,042,430.

CERTIFICATE or common.

May 26. 1936.

BENJAIIIIN enusxm. Y

It is hereby certified that error appears in the printed specificationof the. above numbered patent requiring correction as follows: Page 2,second column, line 1, for "antigent" read antigen; same page andcolumn, line 8', for

"material" read maternal; line 56, for "horus" read hours; and that thesaid Letters Patent should be read with these corrections therein thatthe same may conform to the record of the case in the Patent Office.

Signed and sealed this 21st day of July, A. D. 1936- Henry Van ArsdaleActing Commissioner or Patents.

